Methods have been described for causing two nucleic acid strands to become associated as a result of the presence of a target polynucleotide. These methods are based on forming a noncovalent sandwich involving the target and two probes, each binding to a different site on the target. If the probes are contiguous or separated by one nucleotide, they can be joined in a covalent sandwich by a ligase (Goffin, C. et.al. Nucleic Acids Res. 15(21): 8755 (1987)). The ligated probe can then be amplified using known technology (Saiki, et al., Science 230:1350 (1986)). Regardless of whether the probes are ligated or amplified, provided they are covalently or non-covalently bound, the close association of the two probes can be detected by such known methods as enzyme channeling, fluorescence energy transfer and the like.
Various hybridization methods have been used in order to detect nucleic acid sequences. European Patent Application 0,192,168 describes a solution phase dual hybridization assay for the detection of polynucleotide sequences. The method descibed uses a separation probe which carries a reactive site capable of forming a stable covalent or non-covalent bond with a reaction partner. In the preferred practice of the invention, the reaction partner is attached to a solid support by covalent or non-covalent bonds.
World Patent Application 87/03622 describes a hybridization assay which results in high levels of amplification. Amplification is achieved by taking a primary probe, a small segment of which is hybridized to the target DNA of interest and introducing a second probe which recognizes a separate segment of the target. Using the dual probe system, increased amplification occurs upon the hybridization event taking place.
U.S. Pat. No. 4,775,619 describes a method for the detection of a specific sequence using a hybridization technique such that duplexing of the sample DNA and a probe affects the ability to modify the spatial relationship between a label and a support. The presence of the specific sequence, the target polynucleotide, is determined by the amount of label released into the medium.
U.S. Pat. No. 4,766,062 describes a method for determining the presence of a target polynucleotide in a sample wherein the probe polynucleotide complex is capable of base pair binding such that the target polynucleotide binds to the probe with a displacement of the labeled polynucleotide from the complex. In order for the detection system to be successful there must be sufficient base-pair binding to the target system in order to generate the release of a detectable signal.
Yet another method for the detection of nucleic acid hybridization is described in U.S. Pat. No. 4,724,202. The patent describes a method of detection in which the known sample or separation probe is immobilized on a solid support and contacted with a mixture containing the unknown and a labeled detection probe. The labeled detection probe is created without the use of radioactivity and without chemical modfication by having a single-stranded portion of nucleic acid capable of hybridizing with the unknown connected with a non-hybridizable single or double stranded nucleic acid portion. The non-hybridizable portion includes a recognition site for a particular protein.
A method for detecting the presence of a target nucleotide sequence in a polynucleotide which comprises hybridizing a first nucleotide sequence and a second nucleotide sequence to non-contiguous portions of a target nucleotide sequence and detecting the presence of such first and second nucleotide sequences is set forth in U.S. patent application Ser. No. 07/236,967, now U.S. Pat. No. 5,185,043, the disclosure of which is incorporated herein by reference.
None of the cited background art, however, provide a solution to the problem of detection of nucleic acids as described by the present invention. Using the methods of the present invention, a target polynucleotide sequence can be detected using the solution phase hybridization protocol which may be easily adapted to large scale immunochemical analysis. The methods described in the present invention can be easily applied to the design of diagnostic test systems.